regulation of cellular iron metabolism - biochemical … · regulation of cellular iron metabolism...

Biochem. J. (2011) 434, 365–381 (Printed in Great Britain) doi:10.1042/BJ20101825 365

REVIEW ARTICLERegulation of cellular iron metabolismJian WANG*† and Kostas PANTOPOULOS*†‡1

*Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, U.S.A., †Lady Davis Institute for Medical Research, Sir Mortimer B. Davis JewishGeneral Hospital, 3755 Cote-Ste-Catherine Road, Montreal, QC, H3T 1E2, Canada, and ‡Department of Medicine, McGill University, 1110 Pine Avenue West, Montreal, QC, H3A 1A3,Canada

Iron is an essential but potentially hazardous biometal.Mammalian cells require sufficient amounts of iron to satisfymetabolic needs or to accomplish specialized functions. Iron isdelivered to tissues by circulating transferrin, a transporter thatcaptures iron released into the plasma mainly from intestinalenterocytes or reticuloendothelial macrophages. The binding ofiron-laden transferrin to the cell-surface transferrin receptor 1results in endocytosis and uptake of the metal cargo. Internalizediron is transported to mitochondria for the synthesis of haemor iron–sulfur clusters, which are integral parts of severalmetalloproteins, and excess iron is stored and detoxified in

cytosolic ferritin. Iron metabolism is controlled at different levelsand by diverse mechanisms. The present review summarizesbasic concepts of iron transport, use and storage and focuses onthe IRE (iron-responsive element)/IRP (iron-regulatory protein)system, a well known post-transcriptional regulatory circuit thatnot only maintains iron homoeostasis in various cell types, butalso contributes to systemic iron balance.

Key words: ferritin, ferroportin, iron-regulatory protein 1 (IRP1),iron-regulatory protein 2 (IRP2), iron–sulfur cluster (ISC),transferrin receptor (TfR).

BIOCHEMISTRY AND PHYSIOLOGY OF IRON

With minor exceptions, almost all cells employ iron as a cofactorfor fundamental biochemical activities, such as oxygen transport,energy metabolism and DNA synthesis. This is due to theflexible coordination chemistry and redox reactivity of iron,which allow it to associate with proteins and bind to oxygen,transfer electrons or mediate catalytic reactions [1]. However,iron is also potentially toxic, because, under aerobic conditions,it catalyses the propagation of ROS (reactive oxygen species) andthe generation of highly reactive radicals (such as the hydroxylradical) through Fenton chemistry [2]. As iron readily shuttlesbetween the reduced ferrous (Fe2+) and the oxidized ferric (Fe3+)forms, disruption of the cellular redox equilibrium requires onlycatalytic amounts of the metal. The ensuing oxidative stress isassociated with damage of cellular macromolecules, tissue injuryand disease [3,4]. It should also be noted that oxidized Fe3+ ispoorly bioavailable, despite its high abundance, due to limitedsolubility. Thus the acquisition, usage and detoxification of ironpose a considerable challenge to cells and organisms, which haveevolved sophisticated mechanisms to satisfy their metabolic needsand concomitantly minimize the risk of toxicity [5–7].

The vast majority of body iron (at least 2.1 g in humans) isdistributed in the haemoglobin of red blood cells and developingerythroid cells and serves in oxygen transport. Significant amountsof iron are also present in macrophages (up to 600 mg) and in

the myoglobin of muscles (∼300 mg), whereas excess body iron(∼1 g) is stored in the liver [8,9]. Other tissues contain lower,but not negligible, quantities of iron. Mammals lose iron fromsloughing of mucosal and skin cells or during bleeding, but do notpossess any regulated mechanism for iron excretion from the body.Therefore balance is maintained by the tight control of dietary-iron absorption in the duodenum (Figure 1). The uptake of nu-tritional iron involves reduction of Fe3+ in the intestinal lumenby ferric reductases [such as Dcytb (duodenal cytochrome b)]and the subsequent transport of Fe2+ across the apical membraneof enterocytes by DMT1 (divalent metal transporter 1), a mem-ber of the SLC (solute carrier) group of membrane transport pro-teins, also known as SLC11A2. Dietary haem can also be transpor-ted across the apical membrane by a yet unknown mechanism andsubsequently metabolized in the enterocytes by HO-1 (haem oxy-genase 1) to liberate Fe2+. Directly internalized or haem-derivedFe2+ is processed by the enterocytes and eventually exportedacross the basolateral membrane into the bloodstream via thesolute carrier and Fe2+ transporter ferroportin (also known asSLC11A3). The ferroportin-mediated efflux of Fe2+ is coupledby its re-oxidation to Fe3+, catalysed by the membrane-boundferroxidase hephaestin that physically interacts with ferroportin[10], and possibly also by its plasma homologue ceruloplasmin.

Exported iron is scavenged by Tf (transferrin), which maintainsFe3+ in a redox-inert state and delivers it into tissues. The totaliron content of Tf (∼3 mg) corresponds to less than 0.1 % of

Abbreviations used: Abcb, ATP-binding cassette, subfamily B; ALA, 5-aminolevulinic acid; ALAS, ALA synthase; β-APP, β-amyloid precursor protein;BMP, bone morphogenetic protein; c-aconitase, cytosolic aconitase; C/EBPα, CCAAT/enhancer-binding protein α; Cfd1, cytosolic Fe–S cluster-deficientprotein 1; Cul1, Cullin 1; Dcytb, duodenal cytochrome b; DHBA, dihydroxybenzoic acid; DMT1, divalent metal transporter 1; ER, endoplasmic reticulum;FBXL5, F-box and leucine-rich repeat protein 5; FLVCR, feline leukaemia virus, subgroup C, receptor; Grx, glutaredoxin; H, heavy; HIF, hypoxia-induciblefactor; HO-1, haem oxygenase 1; Hpx, haemopexin; IOP1, iron-only hydrogenase-like protein 1; IRE, iron-responsive element; IRIDA, iron-refractoryiron deficiency anaemia; IRP, iron-regulatory protein; ISC, iron–sulfur cluster; CIA, cytosolic ISC assembly; Isu, iron–sulfur cluster scaffold homologue;L, light; Lcn2, lipocalin 2; LIP, labile iron pool; m-aconitase, mitochondrial aconitase; MRCKα, myotonic dystrophy kinase-related Cdc42 (cell divisioncycle 42)-binding kinase α; Nar1, nuclear architecture-related protein 1; Nbp35, nucleotide-binding protein 35; Nfs, nitrogen fixation homologue; NTBI;non-transferrin-bound iron; PCBP1, poly(rC)-binding protein 1; Rbx1, Ring-box 1; ROS, reactive oxygen species; SDH, succinate dehydrogenase; Skp1,S-phase kinase-associated protein 1; SLC, solute carrier; STAT3, signal transducer and activator of transcription 3; Tf, transferrin; TfR, Tf receptor; UTR;untranslated region.

1 To whom correspondence should be addressed (email [email protected]).

c© The Authors Journal compilation c© 2011 Biochemical Society

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366 J. Wang and K. Pantopoulos

Figure 1 Hormonal regulation of iron efflux from duodenal enterocytes and reticuloendothelial macrophages by hepcidin

Enterocytes absorb inorganic or haem iron from the diet and macrophages phagocytose iron-loaded senescent red blood cells, or acquire iron by other mechanisms (see the main text). Both celltypes release Fe2+ into the plasma via ferroportin (Fpn), which is incorporated into apo-Tf following oxidation to Fe3+ via hephaestin (Heph) or ceruloplasmin (Cp). Hepatocytes generate theiron-regulatory hormone hepcidin in response to high iron or inflammatory signals, which inhibits the efflux of iron via ferroportin and promotes its retention within enterocytes and macrophages.

body iron, but it is highly dynamic and undergoes more than tentimes daily turnover to sustain erythropoiesis. The Tf iron pool isreplenished mostly by iron recycled from effete red blood cellsand, to a lesser extent, by newly absorbed dietary iron. Senescentred blood cells are cleared by reticuloendothelial macrophages,which metabolize haemoglobin and haem, and release iron into thebloodstream. By analogy to intestinal enterocytes, macrophagesexport Fe2+ from their plasma membrane via ferroportin, in aprocess coupled by re-oxidation of Fe2+ to Fe3+ by ceruloplasminand followed by the loading of Fe3+ to Tf (Figure 1).

Ferroportin is expressed in many iron-exporting cells, includingplacental syncytiotrophoblasts, and plays a fundamental rolein the release of iron from tissues into the bloodstream, but

also in maternal iron transfer to the fetus. The completedisruption of mouse ferroportin is embryonic lethal, whereas itsconditional inactivation leads to iron retention and accumulationin enterocytes, macrophages and hepatocytes [11].

REGULATION OF SYSTEMIC IRON TRAFFIC

The ferroportin-mediated efflux of Fe2+ from enterocytes andmacrophages into the plasma is critical for systemic ironhomoeostasis. This process is negatively regulated by hepcidin,a liver-derived peptide hormone that binds to ferroportin andpromotes its phosphorylation, internalization and lysosomal


Regulation of cellular iron metabolism 367

degradation [6,12]. Hepcidin is primarily expressed in hepatocytesas a precursor pro-peptide. Pro-hepcidin undergoes proteolyticprocessing to yield a bioactive molecule of 25 amino acidsthat is secreted into the bloodstream. Hepcidin accumulatesfollowing iron intake and under inflammatory conditions,resulting in decreased dietary-iron absorption and iron retentionin macrophages (Figure 1); during infection, this very probablyserves to deprive invading bacteria from iron that is essentialfor growth. Conversely, hepcidin levels drop in iron deficiency,hypoxia or phlebotomy-induced anaemia, and this responsepromotes intestinal iron absorption and iron release frommacrophages. The disruption of hepcidin is associated withsystemic iron overload (haemochromatosis) [13], whereaspathological elevation of hepcidin levels contributes to thedevelopment of the anaemia of chronic disease [14] and accountsfor the phenotype of hereditary IRIDA (iron-refractory irondeficiency anaemia) [15].

The expression of hepcidin is controlled transcriptionally.Basal hepcidin transcription requires C/EBPα (CCAAT/enhancer-binding protein α) [16]. Iron-dependent induction of hepcidinrequires BMP (bone morphogenetic protein) signalling. Irontriggers the expression of BMP6 in the liver [17] and the intestine[18], which is thought to be secreted into the plasma for bindingto a BMP receptor on the surface of hepatocytes. BMP6 signallingleads to phosphorylation of SMAD1/5/8 and translocation ofSMAD4 to the nucleus, where it promotes hepcidin transcriptionupon binding to proximal and distal sites on its promoter. Thismodel is supported by genetic data with BMP6− /− [19,20] andliver-specific SMAD4− /− [21] mice, which develop iron overloadand express inappropriately low hepcidin levels. It has also beenproposed that hepcidin responds to increased Tf saturation [22],possibly by a mechanism requiring a cross-talk between BMP andMAP (mitogen-activated protein kinase) signalling [23]. Furthercofactors are required for iron-dependent activation of hepcidin,even though their exact mode of action is not yet clear. Theseinclude the haemochromatosis protein HFE, TfR2 (Tf receptor2) and the BMP co-receptor HJV (haemojuvelin). Mutations inthese proteins impair hepcidin expression and lead to hereditaryhaemochromatosis [13]. The membrane-bound serine proteasematriptase 2 (TMPRSS6) appears to inhibit signalling to hepcidinby degrading HJV [24]. Mutations in matriptase 2 are associatedwith IRIDA, which is caused by hepcidin overexpression [15].

The pro-inflammatory cytokine IL-6 (interleukin-6) induceshepcidin transcription via STAT3 (signal transducer and activatorof transcription 3) phosphorylation and translocation to thenucleus for binding to a proximal promoter element [25].IL-1β activates hepcidin via the C/EBPα and BMP/SMADpathways [26]. In addition, ER (endoplasmic reticulum)stress turns on hepcidin transcription via CREBH (cyclicAMP response element-binding protein H) [27] and/or CHOP(C/EBP homologous protein) [28]. Lipopolysaccharide promotesautocrine activation of hepcidin in macrophages via TLR4 (Toll-like receptor 4) signalling [29], whereas the pathogen Borreliaburgdorferi activates myeloid hepcidin via TLR2 [30].

Hepcidin transcription is suppressed during anaemia bya mechanism that requires erythropoietic activity [31]. Inthalassaemic patients, hepcidin expression is blocked uponinduction of GDF15 (growth differentiation factor 15) [32],a member of the TGF-β (transforming growth factor β)superfamily. Likewise, the erythroid regulator TWSG1 (twistedgastrulation homologue 1) was reported to contribute to hepcidinsuppression in cell-culture experiments and in thalassaemic mice[33]. EPO (erythropoietin) signalling downregulates hepcidinfollowing decreased binding of C/EBPα to its promoter [34].Hepcidin transcription is also suppressed by hypoxia and

oxidative stress. The role of HIFs (hypoxia-inducible factors)in the hypoxic pathway of hepcidin is debatable [35], whereasoxidative stress promotes histone deacetylation and decreasedbinding of C/EBPα and STAT3 to the hepcidin promoter [36].

There is no doubt that hormonal regulation of iron efflux fromcells via the hepcidin/ferroportin axis is of paramount importancefor systemic iron homoeostasis. However, it should be noted thatthe expression of ferroportin is also subjected to transcriptional[37] and post-transcriptional (see below) control. Recent resultsshowed that hepcidin-dependent degradation of ferroportindoes not suffice to limit dietary-iron absorption in mice lackingintestinal H-ferritin, adding further complexity to the mechanismsfor regulation of systemic iron homoeostasis [38]. Along similarlines, dietary-iron absorption can be induced independently ofthe hepcidin pathway by transcriptional activation of DMT1and Dcytb in duodenal enterocytes, a response orchestrated byHIF-2α [39,40].

MECHANISMS FOR CELLULAR IRON UPTAKE

The Tf cycle

Developing erythroid cells, as well as most other cell types,acquire iron from plasma Tf. Iron-loaded holo-Tf binds with highaffinity to TfR1 on the surface of cells [41], and the complexundergoes endocytosis via clathrin-coated pits (Figure 2). Aproton pump promotes acidification of the endosome to pH 5.5,triggering the release of Fe3+ from Tf that remains bound toTfR1. The ferrireductase Steap3 reduces Fe3+ to Fe2+ [42], whichis transported across the endosomal membrane by DMT1 to thecytosol or, possibly, directly to mitochondria in erythroid cells[43]. Following the release of iron, the affinity of Tf to TfR1 drops∼500-fold, resulting in its dissociation. In the final step of thecycle, apo-Tf is secreted into the bloodstream to recapture Fe3+.

The importance of the Tf cycle for iron delivery into erythroidcells is underscored by the embryonic lethal erythropoieticdefects associated with the targeted disruption of mouse TfR1,and the microcytic hypochromic anaemia of TfR1+/− animals[44]. Moreover, hpx (hypotransferrinaemic) mice that fail toexpress appropriate Tf levels due to a splicing defect alsodevelop microcytic hypochromic anaemia, which is associatedwith paradoxical hepatic iron overload [45]. These findingsprovide strong evidence that the Tf cycle is indispensable foriron delivery to erythroid cells, and Tf is the only physiologicaliron donor for erythropoiesis.

DMT1 does not only account for dietary-iron absorption inthe intestine, but also constitutes a crucial component of theTf cycle by mediating Fe2+ transport across the endosomalmembrane. Thus DMT1− /− mice can neither absorb dietaryiron, nor efficiently use iron for erythropoiesis and, consequently,develop severe and fatal microcytic hypochromic anaemia shortlyafter birth [46]. Animals with partially inactivated mutant DMT1(mk/mk mice and Belgrade rats) exhibit severe anaemia [47,48].

Other mechanisms for iron uptake

Macrophages may use the Tf cycle for iron uptake, especiallyin culture. However, in vivo, resident macrophages acquire highamounts of iron by phagocytosis of effete red blood cells (Fig-ure 1). Macrophages also have the capacity to clear haemoglobinand haem that is released in the circulation during intravascularhemolysis. Free haemoglobin is scavenged by Hp (haptoglobin), aliver-derived plasma protein, which in turn is internalized by mac-rophages upon binding to the receptor CD163 [49]. Likewise, freehaem is scavenged by plasma Hpx (haemopexin) and undergoes



Figure 2 Cellular iron uptake via the Tf cycle

Iron-loaded holo-Tf binds to TfR1 on the cell surface and the complex undergoes endocytosis via clathrin-coated pits. A proton pump acidifies the endosome, resulting in the release of Fe3+, which issubsequently reduced to Fe2+ by Steap3 and transported across the endosomal membrane to the cytosol by DMT1. Internalized iron is directed to mitochondria via mitoferrin for metabolic utilization(such as synthesis of haem and ISCs), and excess iron is stored in ferritin. A cytosolic fraction of redox-active intracellular iron constitutes the LIP. The apo-Tf–TfR1 complex is recycled to the cellsurface, where apo-Tf is released to capture plasma Fe3+.

endocytosis in macrophages upon binding to the receptorCD91 [50]. Directly internalized haem, or haem derived fromthe degradation of haemoglobin following direct haemoglobinuptake or erythrophagocytosis, is degraded by HO-1, yieldingFe2+, which is released into the plasma through ferroportin.Nramp1 (natural resistance-associated macrophage protein 1),a homologue of DMT1, contributes to efficient recycling ofhaemoglobin iron, especially during haemolytic anaemia [51].

Plasma Tf delivers iron to all tissues, except those that areseparated from the blood by layers of endothelial cells forminga physical barrier (for example the brain, the testis or the eye).How iron crosses the blood–brain and other barriers, which do notallow the free passage of proteins and metabolites, is incompletelyunderstood. It appears that the endothelial cells at the luminal siteof blood capillaries express TfR1 and take up iron from plasmaTf [52,53]. The release of iron from endothelial cells (but alsoneurons or astrocytes) into the brain interstitium very probablyinvolves ferroportin in conjunction with a ferroxidase activity,by analogy to the mechanism for basolateral iron transportin the intestinal epithelium. The ferroxidase activity can beprovided by ferroportin-interacting proteins such as hephaestin,GPI (glycosylphosphatidylinositol)–ceruloplasmin (a membrane-associated ceruloplasmin isoform) [54] or β-APP (β-amyloidprecursor protein) [55]. Inside the interstitial fluid, iron is capturedby brain Tf that is secreted from oligodendrocytes.

Previously, plasma Tf was believed to be spared fromsubstantial glomerular filtration in the kidney [56]. Later, itwas established that polarized epithelial cells from the renal

proximal tubules may acquire Tf-bound iron via the endocyticreceptor cubilin, which operates in conjunction with its co-receptor megalin [57]. Cubilin (but not TfR1) is expressed onthe apical (urine-facing) site of proximal tubule cells, whichalso contain high levels of endosomal DMT1, suggesting thatat least a fraction of Tf is filtered in the kidney and retrievedfrom the glomerular filtrate upon binding to cubilin [56]. It isconceivable that the efflux of iron from the basolateral site of theproximal tubule cells to the bloodstream may involve ferroportinand hephaestin/ceruloplasmin.

Lcn2 (lipocalin 2), a mammalian protein that was identified byits capacity to interact with the bacterial siderophore enterobactin[58], is involved in iron-uptake pathways that operate duringkidney development [59] and injury [60], and under inflammatoryconditions [61]. Iron-loaded Lcn2 is internalized by the receptors24p3R [61] and megalin [62]. Siderophores are low-molecular-mass iron-chelating metabolites, known to be synthesizedby bacteria and fungi to acquire extracellular iron. It wasrecently demonstrated that mammals synthesize the siderophore2,5-DHBA (dihydroxybenzoic acid), which is isomericto 2,3-DHBA, the iron-binding component of enterobactin [63].Importantly, depletion of 2,5-DHBA deregulated iron metabolismin mammalian cells and zebrafish embryos, highlighting thebiological importance of Lcn2-dependent mechanisms. Lcn2− /−

mice exhibit apparently normal iron metabolism; however, theyfail to mount efficient innate immune responses against bacterialinfection, suggesting that Lcn2 serves to deprive bacteria ofnutritional iron [64].



Under physiological conditions, plasma Tf is hyposaturated(to ∼30%) and displays a very high iron-binding capacity, topreclude the accumulation of unshielded NTBI (non-transferrin-bound iron). However, in hereditary haemochromatosis and otheriron-overload disorders, the levels of plasma iron exceed thesaturation capacity of Tf, and a pool of NTBI builds up thatcontributes significantly to hepatic iron loading [65]. The exactchemical nature of NTBI remains elusive, but its redox reactivityand toxicity is well established. It may consist of Fe3+ looselychelated by albumin or by small organic molecules, such ascitrate [66]. The mechanisms of NTBI uptake by cells are poorlyunderstood, but a role for Lcn2 has been excluded [67]. The zinctransporter Zip14 (also known as SLC39A) appears capable ofmediating NTBI uptake [68] as well as promoting the assimilationof Tf-bound iron [69].

Ferritin, the iron-storage protein (see below) has also beenimplicated in iron-transport pathways, which may predominatein pathological states, where iron-rich intracellular ferritin isreleased from damaged tissues. The membrane-bound proteinTIM-2 (T-cell immunoglobulin-domain and mucin-domain 2)functions as a ferritin receptor in rodents [70]. More recently,it was shown that ferritin can be taken up by Scara 5 (scavengerreceptor 5) and donate iron to the developing kidney [71], whereasH-ferritin can be internalized by cells upon specific binding toTfR1 [72].

CELLULAR IRON METABOLISM

Iron usage in mitochondria

Cells mostly use iron in mitochondria for the synthesis ofhaem and ISCs (iron–sulfur clusters). The mechanism forintracellular iron trafficking to mitochondria is incompletelyunderstood. Kinetic evidence and microscopy studies in erythroidcells have provided support for the ‘kiss and run’ hypothesis,which postulates the direct delivery of Tf-derived iron tomitochondria through a transient contact with the endosome[43]. In other cell types, it is generally accepted that ironacquired during the Tf cycle is first released into the cytosoland then targeted to the mitochondria. The conserved cytosolicglutaredoxins Grx3 and Grx4 appear to play an essential rolein intracellular iron sensing and trafficking, as their depletion inyeast leads to impaired iron transport to mitochondria anddefects in iron-dependent pathways [73]. It is also wellestablished that the entry of iron into mitochondria requiresthe SLC transporter mitoferrin (also known as SLC25A37),which is localized to the inner mitochondrial membrane [74].Mitoferrin-1 is highly enriched in erythroid cells and isstabilized during differentiation and following interaction withthe erythroid-specific ABC transporter Abcb10 (ATP-bindingcassette, subfamily B, member 10) [75], whereas mitoferrin-2is ubiquitously expressed and its half-life is not regulated [76].Both mitoferrin isoforms are homologous; however, they exhibitlimited functional redundancy, as overexpression of mitoferrin-2cannot support mitochondrial iron assimilation in erythroid cellsdeficient of mitoferrin-1. The lack of functional mitoferrin-1in the frs (frascati) zebrafish mutant is associated with severedefects in erythropoiesis, haem synthesis and ISC biogenesis [74].Interestingly, depletion of the mammalian siderophore 2,5-DHBAalso leads to defects in mitochondrial iron assimilation and haemsynthesis [63], underscoring a role for the Lcn2 pathway in irondelivery to mitochondria.

All organisms synthesize the tetrapyrrol porphyrin ring ofhaem from the universal precursor ALA (5-aminolevulinicacid) by a conserved eight-step enzymatic pathway [77–79].

Most eukaryotes (except plants), generate ALA in mitochondriaby the condensation of succinyl-CoA and glycine, whichis catalysed by ALAS (ALA synthase). Mammals expressa housekeeping (ALAS1) and an erythroid-specific (ALAS2)isoform of this enzyme. The universal precursor ALA is exportedto the cytosol and converted into the intermediate metabolitesporphobilinogen, hydroxymethylbilane, uroporphyrinogen IIIand coproporphyrinogen III. The latter is oxidized toprotoporphyrinogen IX and imported into the mitochondria,where it is further oxidized to protoporphyrin IX. In thefinal reaction of the haem biosynthetic pathway, ferrochelatasecatalyses the insertion of Fe2+ into protoporphyrin IX. Newlysynthesized haem is exported to the cytosol for incorporationinto haemoproteins. The transport of haem and its metabolicintermediates across the mitochondrial membranes may involvemembers of the ABC family of transporters [77] and the SLCtransporter SLC25A39 [80]. In non-erythroid cells, the rate-limiting step of the haem biosynthetic pathway is the synthesis ofALA. By contrast, the synthesis of the porphyrin ring in erythroidcells depends on the iron supply, which is rate-limiting [79].

The assembly and repair of ISCs is mediated by complexpathways. The functions of several proteins of these pathwayshave been defined, yet new players continue to emerge [81–83].The mitochondrial proteins Isu1/2 (also known as ISCU) andIsa1/2 (also known as ISCA1/2) provide a scaffold for theearly steps of ISC biogenesis. The cysteine desulfurase Nfs1(nitrogen fixation homologue 1, also known as ISCS), incomplex with ISD11 (iron–sulfur protein biogenesis, desulfurase-interacting protein 11), generates elemental sulfur, whereas theiron-binding protein frataxin appears to serve as an iron donor. Themitochondrial proteins Grx5 and the transporter Abcb7 participatein the maturation of ISCs; however, their mode of action isnot clear. The biogenesis of cytosolic ISC proteins requiresmitochondria-derived ISC precursors, which are processed bya dedicated CIA (cytosolic ISC assembly) machinery [82]; todate, various CIA components have been identified, such as theP-loop NTPase Cfd1 (cytosolic Fe–S cluster-deficient protein1), Nar1 (nuclear architecture-related protein 1)/IOP1 (iron-onlyhydrogenase-like protein 1), Cia1, Dre2 and Tah18. Cfd1 interactswith Nbp35 (nucleotide-binding protein 35) and the complex hasbeen proposed to function as a scaffold for cytosolic ISC assembly[84]. An alternative model postulates that the cytosol is equippedwith a complete machinery for de novo ISC assembly, consistingof cytosolic orthologues of mitochondrial ISC assembly factorssuch as Isu/ISCU and Nfs1/ISCS [83]. Importantly, defects inISC biogenesis are associated with deregulation of cytosolic andcellular iron metabolism [43,83].

Handling of excess iron

Cells may eliminate excess intracellular iron by secretion of Fe2+

via ferroportin or by secretion of haem through the putative haemexporter FLVCR (feline leukaemia virus, subgroup C, receptor)[85]. Cells can also store and detoxify excess intracellular ironin the cytosol within ferritin, a conserved protein consisting of24 H (heavy) and L (light) subunits, encoded by distinct genes[86]. Ferritin assembles into a shell-like structure with a cavityof ∼80 Å (1 Å = 0.1 nm) that provides storage space for upto 4500 Fe3+ ions in form of ferric oxy-hydroxide phosphate.Iron may enter ferritin with the aid of PCBP1 [poly(rC)-bindingprotein 1], which appears to function as an iron chaperone[87]. The incorporation of iron into holo-ferritin also requiresthe ferroxidase activity of H-ferritin, whereas L-ferritin chainsprovide a nucleation centre. The levels of H-ferritin and L-ferritindiffer among various tissues; the former is enriched in the heart



and the latter in the liver. Intracellular iron deposits may also bedetected within haemosiderin, a structure consisting of ferritindegradation products and iron oxide clusters.

Iron stored within ferritin is considered to be bioavailableand may be mobilized for metabolic purposes during itslysosomal turnover [88] and, possibly, also following dynamicstructural rearrangements of the ferritin subunits [89]. However,ferritin failed to donate iron for endogenous haem synthesis inexperiments with cultured macrophages [90]. The induction offerroportin promotes mobilization and export of ferritin-derivediron, followed by mono-ubiquitination and degradation of apo-ferritin by the proteasome [91]. Thus ferritin can be degradedby two different pathways, the lysosomal and the proteasomalpathways, which appears to require prior depletion of its iron [92].The iron-storage function of ferritin is crucial for health. Thus theablation of the gene encoding H-ferritin leads to early embryoniclethality [93], whereas the conditional disruption of this genepromotes liver damage due to oxidative stress [94]. Moreover,the intestinal disruption of H-ferritin misregulates dietary-ironabsorption [38]. On the other hand, mutations in the gene encodingL-ferritin are associated with neuroferritinopathy, a rare hereditarymovement disorder with an autosomal dominant transmissionpattern, characterized by accumulation of redox-active iron inthe brain [95].

Mitochondria contain a nuclear-encoded ferritin isoform [96].Mitochondrial ferritin derives from an unusual intronless geneand is synthesized in the cytosol as a precursor polypeptide that istargeted to mitochondria by an N-terminal leader sequence. Themature protein possesses ferroxidase activity and assembles intofunctional ferritin nanocages. Mitochondrial ferritin is normallyexpressed at low levels and does not appear to have any majorfunction in normal mitochondrial iron utilization. Its expression,however, is significantly induced in iron-loaded ring erythroblasts(sideroblasts) of sideroblastic anaemia patients and may serve asa sink for iron deposition [97].

A secreted glycosylated isoform of predominantly L-ferritincirculates in the bloodstream. It contains very low amounts ofiron, suggesting that it does not play an essential role in iron sto-rage or traffic, but it is used as a clinical marker for body ironstores. Experiments in mice suggested that serum ferritin derivesfrom splenic macrophages and kidney proximal tubule cells [98].

LIP (labile iron pool)

Various cell types contain a transient cytosolic pool of iron,presumably bound to low-molecular-mass intracellular chelates,such as citrate, various peptides, ATP, AMP or pyrophosphate.This LIP is redox-active and can be monitored in situ by usingfluorescent sensors such as calcein or Phen Green SK [99,100].Experiments with other fluorescent sensors that are selective formitochondria also suggest the presence of chelatable iron in thisorganelle [101]. Ferritin and iron-chelating drugs protect cellsagainst oxidative damage by reducing the cytosolic LIP in culturedcells [102] and in animals [103]. However, chronic overexpressionof ferritin may lead to the opposite effect in mice, suggestingthat iron sequestered in the ferritin shells may eventually becomepro-oxidant [104]. The cytosolic LIP mirrors the cellular ironcontent and its fluctuations are considered to trigger homoeostaticadaptive responses.

THE IRE (IRON-RESPONSIVE ELEMENT)/IRP (IRON-REGULATORYPROTEIN 1) REGULATORY SYSTEM

The expression of TfR1 and ferritin are co-ordinately regulatedpost-transcriptionally upon binding of IRP1 or IRP2 to IREs

Figure 3 Typical IRE motif

A typical IRE motif consists of a hexanucleotide loop with the sequence 5′-CAGUGH-3′

(H could be A, C, or U) and a stem, interrupted by a bulge with an unpaired C residue(left) or an asymmetric tetranucleotide bulge (right); the latter is characteristic of ferritin IRE.Base-pairing between C1 and G5 of the loop is functionally important.

in the UTRs (untranslated regions) of their respective mRNAs[105–107]. IREs (Figure 3) are evolutionarily conserved hairpinstructures of 25–30 nt [108]. A typical IRE stem consists ofvariable sequences that form base pairs of moderate stability(�G≈− 7 kcal/mol), and folds into an α-helix that is slightlydistorted by the presence of a small bulge in the middle(an unpaired C residue or an asymmetric UGC/C bulge/loopcommonly found in the ferritin IRE). The loop contains aconserved 5′-CAGUGH-3′ sequence (H denotes A, C or U),where the underlined C and G residues form a base pair. TfR1mRNA contains multiple IREs within its long 3′UTR, whereasthe mRNAs encoding H- and L-ferritin contain a single IRE intheir 5′UTRs.

In iron-starved cells, IRPs bind with high affinity (Kd ≈10− 12

M) to cognate IREs. The IRE–IRP interactions stabilize TfR1mRNA and, moreover, impose a steric blockade to ferritin mRNAtranslation (Figure 4). As a result, increased TfR1 levels stimulateacquisition of iron from plasma Tf to counteract iron deficiency.The inhibition of de novo ferritin synthesis leads to decreasedabundance of this protein, as iron storage becomes obsolete underthese conditions. Conversely, in cells with high iron content, bothIRP1 and IRP2 become unavailable for IRE binding, allowingTfR1 mRNA degradation and ferritin mRNA translation. Thuswhen iron supply exceeds cellular needs, the IRE–IRP switchminimizes further iron uptake via TfR1, and favours the storage ofexcess iron in newly synthesized ferritin. The pathophysiologicalsignificance of the IRE–IRP system is illustrated in HHCS(hereditary hyperferritinaemia–cataract syndrome), an autosomaldominant disorder, characterized by supra-physiological serumferritin levels (without iron overload) and early-onset cataract,that is caused by mutations in L-ferritin IRE that prevent IRPbinding [109].

The IRE–IRP system was initially defined as a relativelysimple and ubiquitous post-transcriptional regulatory circuitthat maintains cellular iron homoeostasis in vertebrates byorchestrating co-ordinated iron uptake by TfR1 and storage inferritin. The identification of additional IRE-containing mRNAsand the ongoing biochemical and physiological characterizationof IRPs added considerable complexity and uncovered a



Figure 4 Post-transcriptional control of cellular pathways by the IRE–IRP regulatory system

Translational-type IRE–IRP interactions in the 5′UTR modulate the expression of the mRNAs encoding H- and L-ferritin, ALAS2, m-aconitase, ferroportin, HIF-2α, β-APP and α-synuclein, whichin turn control iron storage, erythroid iron utilization, energy homoeostasis, iron efflux, hypoxic responses and neurological pathways respectively. Conversely, IRE–IRP interactions in the 3′UTRstabilize the mRNAs encoding TfR1, DMT1, Cdc14A and MRCKα, which are involved in iron uptake, iron transport, the cell cycle and cytoskeletal remodelling respectively. Note that the regulationof DMT1, Cdc14A and MRCKα may require additional factors, and that the IREs in Cdc14A and MRCKα mRNAs are not phylogenetically conserved.

functional significance for the IRE–IRP system that exceeds thenarrow boundaries of cellular iron uptake and storage. Limitationsof this system have also been reported, mostly in specializedcells that may override IRE–IRP-mediated control mechanismsfor efficient iron handling. For example, TfR1 mRNA stabilityis uncoupled from iron supply and IRP regulation in erythroidprogenitor cells, which take up extraordinary amounts of ironfor haem synthesis and haemoglobinization [110]; under theseconditions, TfR1 expression is regulated transcriptionally byan erythroid active element in its promoter [111]. IRE–IRP-independent regulation of TfR1 and ferritin at the transcriptionallevel is well established, and is reviewed elsewhere [112,113].

OTHER IRE-CONTAINING mRNAS

The biochemical characterization of IREs and the establishmentof a canonical IRE motif prepared the way for the discovery offurther IRE-containing mRNAs, some of them bearing atypical,yet functional, IREs [105–107]. An in silico screen of nucleotidedatabases led to the identification of a ‘translation-type’ IRE (anIRE that confers translational regulation) in the 5′UTR of ALAS2

mRNA. Considering that ALAS2 catalyses the initial reaction forhaem biosynthesis in erythroid cells, the translational repressionof its mRNA by IRPs associates the IRE–IRP system withsystemic iron utilization and homoeostasis. This response mayinhibit the accumulation of toxic protoporphyrin IX when iron isscarce. The severe hypochromic anaemia and early embryoniclethality of the sir (shiraz) zebrafish mutant is caused by agenetic defect in Grx5, which impairs the regulation of IRP1 (seebelow) and eventually leads to translational repression of ALAS2[114]. A similar, but relatively milder, biochemical phenotype hasbeen documented in a human patient with sideroblastic anaemia;interestingly, ALAS2 mRNA translation was suppressed by bothIRPs because the deregulation of IRP1 was associated withcytosolic iron depletion and concomitant induction of IRP2 [115].

A single translation-type IRE was also found in the 5′UTR ofthe mRNAs encoding mammalian m-aconitases (mitochondrialaconitases) and the Ip (iron protein) subunit of DrosophilaSDH (succinate dehydrogenase), which are both iron–sulfurenzymes of the citric acid cycle. The latter is not conservedin SDH transcripts of other insects [108]. Quantitatively, thedegree of IRP-mediated translational repression of mammalianm-aconitases and Drosophila SDH is not profound (as compared



with ferritin); however, it may suffice to co-ordinate the expressionof these iron-containing proteins with iron availability and linkthe IRE–IRP system with energy metabolism.

The mRNAs encoding the iron transporters DMT1 andferroportin are expressed in alternatively spliced isoforms, someof which are furnished with a translation-type IRE. Two out offour DMT1 transcripts contain a single IRE in their 3′UTR[116] that presumably operates as a stability control elementand accounts for the high duodenal DMT1 expression in iron-deficient mice [117]. Nevertheless, the function of DMT1 IREappears to be cell-specific and to require additional upstreamregulatory elements in exon 1A [116]. Ferroportin mRNA isexpressed in two alternatively spliced transcripts, one of whichcontains a single translation-type IRE in its 5′UTR [118] thatis consistently associated with high ferroportin expression inthe livers of iron-loaded mice [119]. Conversely, the lack ofIRE in the alternative ferroportin transcript, which is enrichedin duodenal enterocytes and erythroid precursor cells [118],allows the accumulation of ferroportin in these tissues duringiron deficiency [119,120] by evading the translational blockadeimposed by active IRPs. In an iron-deficient state, the bypass ofthe IRE–IRP system contributes to homoeostatic adaptation by(i) probably facilitating dietary-iron absorption in the duodenum,and (ii) possibly also permitting efflux of iron from erythroid cellsin the bloodstream to restrict erythropoiesis and to make the metalavailable to iron-starved non-erythroid cells. The irradiation-induced deletion of 58 bp from mouse ferroportin IRE has beenassociated with diverse phenotypes, ranging from erythropoietin-dependent polycythaemia in heterozygous animals to microcytichypochromic anaemia in homozygous counterparts [121]. Theseresults may indicate further complexity in the post-transcriptionalregulation of ferroportin by the IRE–IRP system.

More recently, a high-throughput biochemical screen revealedan atypical IRE in the 5′UTR of HIF-2α mRNA that functionsas a translational control element [122]. In vitro, HIF-2α IREinteracts efficiently with recombinant IRP1 and IRP2 [122],but it appears to be a preferential target of IRP1 in cells[123,124]. Importantly, HIF-2α regulates hepatic erythropoietinproduction [125] and its ablation is associated with anaemia[126]. Thus translational inhibition of HIF-2α mRNA by IRP1may lead to reduced erythropoietin expression, which can beviewed as a homoeostatic response to curtail erythropoiesis in irondeficiency. Since HIF-2α mediates the transcriptional activation ofduodenal DMT1 and Dcytb in iron deficiency [39,40], its transla-tional regulation by the IRE–IRP system is also predicted to fine-tune dietary-iron absorption. The development of animal modelswith disrupted HIF-2α IRE could provide a valuable tool to clarifythe physiological role of IRPs in HIF-2α regulation.

Other experiments resulted in the identification of single IREmotifs in the 3′UTR of mRNA splice variants encoding MRCKα[myotonic dystrophy kinase-related Cdc42 (cell division cycle42)-binding kinase α] [127] and human Cdc14A phosphatase[127,128]. Preliminary biochemical characterization suggests thatthese IRE motifs contribute to the regulation of mRNA stability,linking the IRE–IRP system with cytoskeletal remodelling andthe cell cycle. Nevertheless, these IREs do not exhibit extensivephylogenetic conservation and appear to be restricted to primatesand some mammals [108]. The mRNA encoding β-APP harboursa non-canonical IRE motif with a conserved 5′-CAGAG-3′

sequence (the underlined C and G residues form a base pair) as partof an extended loop in its 5′UTR, which preferentially interactswith IRP1 and functions as a translational control element[129]. Interestingly, α-synuclein mRNA also contains a predictedIRE-like motif [130] that awaits functional characterization.Aberrant expression of β-APP and α-synuclein is associated with

Alzheimer’s and Parkinson’s diseases respectively; thus validationof the regulatory function of their IREs may couple the IRE–IRPsystem with human neurodegenerative conditions.

An extensive phylogenetic analysis confirmed that IRE-containing mRNAs are exclusively found in metazoans [108].The ferritin IRE motif may represent the ancestral prototype,which was subsequently adopted during evolution by othergenes in higher organisms. Notably, IRE-like sequences withiron-regulatory functions are present in some bacterial mRNAs[131–133]; however, their modes of action are only distallyreminiscent of the metazoan IRE–IRP network.

Overall, as illustrated in Figure 4, functional IRE motifs havethus far been identified in mRNAs encoding proteins of ironuptake (TfR1), storage (H- and L-ferritin), erythroid utilization(ALAS2) and transport (DMT1 and ferroportin), as well asenergy metabolism (m-aconitase and Drosophila SDH), hypoxicregulation (HIF-2α), cytoskeletal reorganization (MRCKα), cell-cycle control (Cdc14A) and neuronal function (β-APP andα-synuclein). It is also worth noting that the mRNAs encodingTfR2, mitochondrial ferritin and ALAS1 do not possess an IRE.The expanded list of IRE-containing mRNAs emphasizes the roleof the IRE–IRP system as a master post-transcriptional iron-regulatory switch, but also implies further regulatory potentialoutside the context of iron metabolism in a strict sense.

IRPs: FUNCTIONAL AND STRUCTURAL FEATURES

IRP1 and IRP2 do not share sequence similarities with knownRNA-binding proteins and do not contain any established RNA-binding motifs. They both belong to the family of ISC isomerases,which includes m-aconitase [105–107]. This enzyme catalysesthe isomerization of citrate to iso-citrate via the intermediatecis-aconitate during the citric acid cycle, and contains a cubane[4Fe–4S]2+ ISC in its active site. Three of the iron atoms areattached to cysteine residues of the polypeptide, whereas thefourth iron (Fea) remains free and mediates catalytic chemistry.

IRP1 assembles an analogous to m-aconitase ISC that convertsit to a c-aconitase (cytosolic aconitase). However, in contrast withm-aconitase, IRP1 only retains its ISC and its enzymatic functionin iron-replete cells. In iron deficiency, holo-IRP1 is converted intoapo-protein that possesses IRE-binding activity. Thus IRP1 isbifunctional and its mutually exclusive activities are reversiblyregulated by an unusual ISC switch. IRP1 probably evolvedindependently of m-aconitase following an early duplication eventthat allowed it to acquire IRE-binding activity [134]. A secondduplication event led to the evolution of IRP2 in higher eukaryotes.

IRP2 shares extensive homology with IRP1; however, it neitherassembles an ISC nor retains aconitase active-site residues.Consequently, IRP2 only exhibits an IRE-binding activity anddoes not have any enzymatic function. A feature of IRP2 thatdistinguishes it from IRP1 is the presence of a conserved cysteine-and proline-rich stretch of 73 amino acids close to its N-terminus.This sequence is encoded by a separate exon and appears to beunstructured [135]. IRP2 is regulated in an irreversible manner,at the level of protein stability.

The crystal structure of IRP1 has been solved in both thec-aconitase-binding [136] and IRE-binding [137] forms(Figure 5), although the structure of IRP2 has not yet beendetermined. These results have validated earlier evidence thatthe site for catalysis and RNA-binding overlap, and the switchbetween the enzymatic and RNA-binding forms is associatedwith extensive conformational rearrangements. The folding ofholo-IRP1 follows the pattern of m-aconitase [136], despiterelatively limited sequence identity (22 %), but consistently with



Figure 5 Crystal structure of IRP1

(A) c-Aconitase form (PDB code 2B3X); (B) IRE-binding form (PDB code 2IPY). A three-dimensional structure of this Figure is available at http://www.BiochemJ.org/bj/434/0365/bj4340365add.htm.

the conservation of active-site residues. The protein is composedof four globular domains. Domains 1–3 are compact and joindomain 4 through a surface linker. The ISC is located centrallyat the interface of the four domains. The topology of the ISC andthe surrounding environment are fairly conserved between c- andm-aconitases. Nevertheless, the overall structure of holo-IRP1,a protein of 889 amino acids, also shows differences to that ofm-aconitase, which is smaller (780 amino acids). The short IRP1fragments that do not superimpose with m-aconitase are exposedon the surface of the protein. As a result, the shapes and surfacetopologies of holo-IRP1 and m-aconitase diverge substantially,which may explain the fact that only the former can acquire IRE-binding activity.

The structure of IRP1 in a complex with ferritin IRE (at 2.8 Åresolution) uncovered the details of the protein reorganizationupon loss of its ISC [137]. The main features are a rotation ofdomain 4 by 32 ◦, but also an unpredicted extensive rearrangementof domain 3 by 52 ◦ that creates a hydrophilic cavity and allowsaccess to the IRE. The RNA–protein interaction requires twocrucial segments at the interface of domain 2 (residues 436–442)and domain 3 (residues 534–544). Thr438 and Asn439 make directcontacts with the IRE. The terminal residues of the IRE loop, A15,G16 and U17, interact with Ser371, Lys379 and Arg269 respectivelywithin a cavity between domains 2 and 3. A second bindingsite is formed around the unpaired-C-bulge residue between theupper and lower stem, which occupies a pocket within domain 4,sandwiched between Arg713 and Arg780. The IRE–IRP1 complexis stabilized by additional bonds, ionic interactions and van derWaals contacts.

The structural studies described above offered detailed insightsinto the dual function of IRP1 as a c-aconitase and an IRE-bindingprotein. These findings also enabled the modelling of IRP2structure and the design of site-directed mutagenesis experimentsto investigate the role of single amino acid residues of IRP2 inIRE binding [138]. Nevertheless, the crystallization of IRP2 andthe resolution of its structure, especially in a complex with IRE,will be necessary to precisely map the RNA–protein interactionand to understand the topology of the IRP2-specific 73 amino acidinsert and its possible role in IRE binding.

IRON-DEPENDENT REGULATION OF IRP1

The ISC of IRP1 is the major site for its regulation (Figure 6).In vitro, holo-IRP1 can be easily reconstituted upon incubationof apo-IRP1 with ferrous salts, sulfide and reducing agents[139]. Within cells, the conversion of apo- to holo-IRP1 requiresseveral cofactors, such as the mitochondrial proteins Nfs1 (ISCS)[140–142], frataxin [143,144], ISCU [145], Grx5 [114,115],ISD11 [146] or Abcb7 [147], as the silencing of these proteinsfavours expression of IRP1 in the IRE-binding form due toimpaired ISC biogenesis. These results support an active roleof mitochondria in the assembly of holo-IRP1. Moreover,mitochondrial ATP production is required for maintenance andrepair of the IRP1 ISC [148], and ATP binds directly to IRP1and promotes ISC stabilization [149]. Cytosolic homologues ofIsu/ISCU and Nfs1/ISCS [145,150], as well as the cytosolicproteins Cfd1 [151], Nbp35 [152], Nar1/IOP1 [153], Cia1 [154],Dre2 [155] and, possibly, Tah18, which transfers electrons to Dre2[156], are also implicated in the ISC assembly of IRP1.

Iron starvation leads to conversion of holo-IRP1 into an IRE-binding apo-protein following depletion of its ISC. In culturedcells, this process is relatively lengthy (8–12 h) and does notrequire de novo protein synthesis. IRP1 is a fairly stable protein(half-life of ∼24 h) and, under normal circumstances, its stabilityremains unaffected by iron levels. However, when ISC biogenesisis impaired by either inactivation of ISC assembly cofactors orphosphorylation of IRP1 at Ser138, iron leads to ubiquitination andslow degradation of apo-IRP1 by the proteasome [142,157,158].This backup mechanism prevents accumulation of excessive apo-IRP1 in iron-loaded cells that may disrupt iron homoeostasis by itsunregulated IRE-binding activity. Iron-chelating drugs promoteconsiderably more efficient conversion of holo- to apo-IRP1 intypical cell-culture conditions with 21% oxygen, as comparedwith lower oxygen concentrations (3–6%) that are more relevantphysiologically in tissues [159]. This is consistent with thepredominance of holo-IRP1 in tissues of iron-deficient animals,and the conversion of only a small fraction to an IRE-bindingapo-protein. The ISC of IRP1 exhibits sensitivity to oxidants (seebelow), whereas hypoxia favours its stabilization [123,160].


http://www.BiochemJ.org/bj/434/0365/bj4340365add.htm


Figure 6 Under physiological conditions, IRP1 is regulated by a reversible ISC switch

Iron deficiency promotes ISC disassembly and a conformational rearrangement, resulting in the conversion of IRP1 from c-aconitase to an IRE-binding protein. The ISC is regenerated in iron-repletecells. Hypoxia favours maintenance of the ISC, whereas H2O2 or NO promote its disassembly. When the ISC-biogenesis pathway is not operational, iron leads to ubiquitination of apo-IRP1 by theFBXL5 E3 ligase complex (including Skp1, Cul1 and Rbx1), resulting in proteasomal degradation.

IRP1 can be phosphorylated by PKC (protein kinase C) atthe conserved Ser138 and Ser711 residues [139,157,161]. Ser138

is located in proximity to the ISC and its phosphorylationappears to interfere with the ISC stability and alter the set-point for its disassembly in an oxygen-dependent manner [160].The introduction of a phosphomimetic S138E mutation favoursnon-oxidative demetallation of [4Fe–4S]2+ to [3Fe–4S]0 byiron-chelating drugs in vitro [160] and sensitizes IRP1 to iron-dependent degradation in cells [157,158]. Phosphorylation of theC-terminal Ser711 may also regulate IRP1 functions. An S711Esubstitution inhibits the capacity of IRP1 to convert citrate into theintermediate cis-aconitate in vitro [139,161] and impairs its IRE-binding activity in cells [139]. PKC-mediated phosphorylationhas been reported to promote the translocation of IRP1 from thecytosol to the ER and Golgi in iron-deficient cells, probably tofacilitate its interaction with TfR1 IREs [162].

IRON-DEPENDENT REGULATION OF IRP2

IRP2 is synthesized de novo in response to low iron and remainsstable under iron-starvation or hypoxia [105–107]. In iron-

replete cells, however, IRP2 becomes destabilized and undergoesrapid ubiquitination and degradation by the proteasome. Earlierassumptions that the stability of IRP2 is regulated by its specific73 amino acid insert have been experimentally rejected [163,164].The analysis of a series of IRP2 deletion mutants providedevidence that its C-terminus contains sequences that are necessary,but not sufficient, for iron-dependent degradation [165]. Theseresults suggested that the recognition of IRP2 by the proteasomaldegradation machinery may require additional IRP structuralelements. DMOG (dimethyl-oxalyl-glycine), an inhibitor of2-oxoglutarate-dependent oxygenases, partially stabilizes IRP2against iron, suggesting a possible involvement of this familyof enzymes in tagging IRP2 for degradation [163,164]. Similareffects were observed within cells treated with succinylacetone,an inhibitor of haem synthesis, [166–168], highlighting a potentialrole of endogenous haem in the control of IRP2 stability.

More recent results demonstrated that IRP2 (as well as apo-IRP1) are substrates of FBXL5 (F-box and leucine-rich repeatprotein 5), a member of an E3 ubiquitin ligase complex thatalso includes Skp1 (S-phase kinase-associated protein 1), Cul1(Cullin 1) and Rbx1 (Ring-box 1) [169,170]. FBXL5 contains anN-terminal haemerythrin domain with a characteristic Fe–O–Fe



Figure 7 Iron and oxygen-dependent regulation of IRP2 stability by FBXL5

IRP2 is stable in iron deficient and/or hypoxic cells; under these conditions, FBXL5 undergoes ubiquitination and proteasomal degradation. An increase in iron and oxygen levels stabilizes FBXL5by formation of an Fe–O–Fe centre in its haemerythrin domain, triggering the assembly of an E3 ubiquitin ligase complex together with Skp1, Cul1 and Rbx1. This complex ubiquitinates (Ub) IRP2,leading to its recognition by the proteasome and its degradation.

centre. Notably, FBXL5 is the first mammalian protein identifiedto harbour this ancient domain, which is used by oxygen-transport proteins in some bacteria and lower eukaryotes [171].In iron-replete and oxygenated cells, FBXL5 accumulates andinteracts with IRP2, mediating its ubiquitination and subsequentdegradation (Figure 7). In contrast, in iron-deficient or hypoxiccells, FBXL5 itself undergoes proteasomal degradation by a yetunknown mechanism upon the loss of its Fe–O–Fe centre, whichallows the stabilization of IRP2. Deletion of the haemerythrindomain or mutation of iron-binding ligands abolishes theregulatory function of FBXL5. Hence, FBXL5 senses iron andoxygen levels through the Fe–O–Fe centre of its haemerythrindomain and emerges as a novel regulator of cellular ironhomoeostasis.

IRP2 can be phosphorylated at Ser157 by Cdk1 (cyclin-dependent kinase 1)/cyclin B1 during the G2/M-phase of thecell cycle; this response decreases its IRE-binding activity andderepresses ferritin mRNA translation [172]. During mitoticexit, Ser157 undergoes dephosphorylation by the phosphataseCdc14A. The regulation of Cdc14A during the cell cycle wasfound to be iron independent, despite the presence of the IREmotif in one of its mRNA splice variants. The stimulation offerritin synthesis coupled to the inactivation of phosphorylatedIRP2 is thought to protect DNA from iron-catalysed oxidativedamage.

REDOX REGULATION OF IRON METABOLISM

Both IRP1 and IRP2 are sensitive to ROS and RNS (reactivenitrogen species) (reviewed in [173]). The redox regulation of

IRP1 is mediated by its ISC. Exposure of cells to micromolarconcentrations of H2O2 leads to removal of the ISC and inductionof IRP1 for IRE binding via an incompletely characterizedsignalling mechanism. This response can be antagonized bymyeloperoxidase-derived hypochlorite [174]. In vitro, ROS andRNS remove the Fea of the ISC and convert [4Fe–4S]2+-IRP to[3Fe–4S]2+-IRP1, which is non-functional. IRP1 also respondsto NO, which likewise induces IRE binding at the expense ofits aconitase activity, albeit by a diverse mechanism. NO maydestroy the ISC of IRP1, but also promote iron efflux from cells[175], suggesting that the NO-mediated switch of IRP1 to itsIRE-binding form could represent a homoeostatic response toiron starvation.

Consistent with this hypothesis, NO released from transfectedfibroblasts was found to stabilize IRP2 in co-cultured cells[176]. The first hints for redox regulation of IRP2 wereprovided by experiments showing that antioxidants, suchas ascorbate, α-tocopherol and N-acetylcysteine promote theproteasomal turnover of IRP2 [164]. More recently, it was shownthat oxidants stabilize previously induced IRP2 against iron-dependent degradation [176a]. On the other hand, ROS may alsooxidize Cys512 and Cys516, predicted to lie within the IRE-bindingcleft of IRP2. Formation of a disulfide bridge between these twocysteine residues was shown to inhibit the IRE-binding activityof IRP2 and reduce TfR1 mRNA abundance [138].

The well documented induction of IRP1 by oxidative stressmodulates cellular iron metabolism. Thus H2O2 leads totransient inhibition of ferritin synthesis, up-regulation of TfR1,increased uptake of 59Fe-labelled Tf and, notably, elevatedstorage of 59Fe into ferritin, despite the decrease in the ferritin



content [177]. Oxidative stress may also regulate cellulariron metabolism independently of IRPs, as sustained non-toxic H2O2 concentrations (<5 μM), which mimic inflammatoryconditions and do not affect IRE-binding activity, stimulateTfR1 mRNA translation [178]. Moreover, higher H2O2 dosespromote proteasomal degradation of ferritin [179]. Importantly,ROS stabilize HIF-α subunits by oxidizing Fe2+ to Fe3+ andthereby depleting an essential cofactor of prolyl hydroxylases,which initiate their degradation in normoxia [180]. This hasprofound implications for iron metabolic pathways, consideringthat HIF transcriptionally regulates DMT1, Dcytb, Tf, TfR1,ceruloplasmin, HO-1 and ALAS2 [39,181,182]. The ROS-dependent repression of hepcidin via C/EBPα [36,183,184]further highlights the multitude of pathways for redox regulationof iron metabolism.

PHYSIOLOGICAL FUNCTIONS OF IRP1 AND IRP2

Targeted disruption of both IRP1 and IRP2 is incompatible withlife and leads to embryonic lethality at the blastocyst stage[185], illustrating the importance of these proteins in earlydevelopment. Mice with tissue-specific ablation of both IRP1and IRP2 have been generated by Cre/loxP technology. In theintestine, the lack of IRPs is associated with growth defects, in-testinal malabsorption, dehydration, weight loss and death within4 weeks of birth [186]. The deregulated expression of TfR1,ferritin, ferroportin and DMT1 is consistent with a critical functionof IRPs in intestinal iron transport and storage. The lack ofIRPs in hepatocytes also leads to premature death due to liverfailure, mitochondrial disfunction and alterations in haem andISC biosynthetic pathways [187].

Single IRP1− /− or IRP2− /− mice are viable, indicating afunctional redundancy of the ubiquitously expressed IRP1 andIRP2. Nonetheless, it appears that IRE-containing mRNAs areprimarily regulated by IRP2 in vivo, as IRP1 is predominantlyexpressed as c-aconitase within tissues [105,188]. Hence, it isnot surprising that IRP1− /− mice do not exhibit any overtpathological phenotype under standard laboratory conditions,and merely misregulate TfR1 and ferritin expression in thekidney and brown fat [189]. IRP1− /− mice also mount anefficient inflammatory signalling response to turpentine [190] anddo not exhibit differential sensitivity to anthracycline-inducedcardiotoxicity compared with wild-type animals [191]. Thelack of any discernible pathology in IRP1− /− mice may raisequestions on the physiological significance of IRP1 as c-aconitase.Considering the important roles of citrate in metabolism [192],it will be important to examine IRP1− /− animals for potentialmetabolic defects.

IRP2− /− mice develop microcytic hypochromic anaemia,manifest excessive iron deposition in the duodenum and theliver and have relative iron deficiency in the spleen [193,194].Erythroid precursor cells of IRP2− /− animals exhibit reducedTfR1 expression, which may account for the decreased ironlevels in the bone marrow. These cells also contain high levelsof protoporphyrin IX, apparently due to unrestricted expression ofALAS2, which is reminiscent of erythropoietic protoporphyria.The characterization of mice with tissue-specific disruption ofIRP2 in either enterocytes, hepatocytes or macrophages showedthat the deregulation of tissue iron levels during ubiquitous IRP2deficiency is largely explained by cell-autonomous functionsof IRP2 [195]. Thus selective ablation of IRP2 in enterocytes,hepatocytes or macrophages impairs iron homoeostasis in thesecells, but does not alter haematological or plasma iron parameters,suggesting that the microcytosis of IRP2− /− mice is caused byan intrinsic defect in haematopoiesis.

The disruption of IRP2 has also been associated with neuropath-ology. Aging IRP2− /− mice accumulate excessive amounts ofiron in specific areas of the brain and develop a progressive neuro-degenerative disorder [196,197]. This clinical phenotype is furtheraggravated in a genetic background of IRP1 haploinsufficiency(IRP2− /− IRP1+/− mice) [198]. Treatment of IRP2− /− mice withthe ISC-disrupting nitroxide Tempol partially rescued neuropathyin these animals, due to activation of endogenous IRP1 for IREbinding [199]. However, Tempol failed to correct microcytosis,possibly due to lower expression of IRP1 in erythroblasts com-pared with the forebrain. Interestingly, isogenic IRP2− /− micegenerated by a different targeting strategy do not manifest severeneurodegeneration, but perform relatively poorly in neurobehav-ioural tests [200]. The role of IRP2 in neuronal physiology isunclear. It has been hypothesized that disruption of iron homoeo-stasis in neurons of IRP2− /− animals (which show suppressionof TfR1 and unrestrained expression of ferritin and, possibly, alsoferroportin) may cause a state of functional iron deficiency [105].In addition, the lack of IRP2 may trigger a decrease in braincopper levels by derepressing translation of the IRE-containingβ-APP mRNA, which is also implicated in copper efflux [201].

Taken together, the results obtained from the analysis ofcurrently available IRP knockout mice demonstrate an essentialrole of the IRE–IRP system in body iron homoeostasis. Furthercharacterization of these animals, as well as the generation of newmodels, e.g. bearing spatial and/or temporal disruption of IRPs, orengineered for tissue-specific expression of constitutive mutants,is expected to improve our understanding on the function of theIRE–IRP network in health and disease.

IRPs AND CANCER

There is evidence for possible involvement of IRPs in cancerbiology. The overexpression of wild-type IRP1 or IRP1C437S, amutant with constitutive IRE-binding activity, in human H1299lung cancer cells did not affect their proliferation in culture, butsignificantly impaired the growth of tumour xenografts formedupon injection of these cells into nude mice [202]. In contrast, ina similar setting, the overexpression of IRP2 strongly acceleratedtumour xenograft growth, and this apparent pro-oncogenicactivity was abolished by deletion of the IRP2-specific domain of73 amino acids [203]. Interestingly, these contrasting phenotypeswere not associated with differential expression of known IRPtargets. In fact, ferritin evaded the suppressor activity of bothIRP1 and IRP2 transgenes in the tumour xenografts, as previouslyshown in dense cultures of IRP1C437S-overexpressing cells [204].IRP1- and IRP2-overexpressing tumours exhibited distinct gene-expression profiles, suggesting that IRPs may differentiallymodulate cancer growth by mechanisms independent of theirfunction as IRE-binding proteins. The pro-oncogenic activityof IRP2 correlated with increased ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation and high levels ofc-myc, which is particularly intriguing considering that this proto-oncogene transcriptionally activates IRP2 [205]. Further workis required to understand the molecular basis underlying theapparent tumour suppressor activity of IRP1 and to elucidatewhether IRP2 functions as a bona fide oncogene and, possibly,participates in a feedback regulatory loop with c-myc to controltumour growth.

PERSPECTIVES

Until the mid-1980s, the bio-iron field was restricted to thestudy of Tf, TfR1 and ferritin. The subsequent discovery of



the IRE–IRP system prepared the way for understanding theco-ordinate regulation of cellular iron metabolism. In the newmillennium, the identification of hepcidin as a master hormonalswitch of systemic iron homoeostasis has provided a framework tocomprehend the regulation of physiological mechanisms for irontraffic in the body and to decipher the molecular basis underlyingvarious iron-related disorders. Today, an in-depth characterizationof the signalling pathways and cross-talk activities culminating inthe activation of hepcidin continues to pose a major challenge.This includes elucidation of the early responses to systemiciron perturbations or alterations in erythropoietic activity thattranslate into hepcidin regulation. It should also be noted that theprofound biomedical implications associated with the modulationof hepcidin pathways render the hepcidin–ferroportin axis anattractive target for drug development.

It will be important to further characterize and betterunderstand the regulatory links between systemic and cellulariron metabolism. For example, the post-transcriptional regulationof ferroportin and HIF-2α by IRPs is of particular interest,considering that the former is the target of hepcidin, whereas thelatter is an upstream regulator of erythropoiesis and intestinal ironabsorption. Moreover, TfR1, a target of IRPs, is crucial for iron-dependent regulation of hepcidin. Future work is also expectedto clarify the role of the IRE–IRP system in neuronal biology,in light of the biochemically characterized non-canonical IREand the predicted IRE in the mRNAs encoding β-APP and α-synuclein respectively. It is also conceivable that additional IRE-containing mRNAs, especially with atypical IRE motifs, remainto be discovered. Although studies of IRP knockout animals haveprovided crucial insights into the role of the IRE–IRP regulatorysystem in vivo, the physiological significance of an IRE motifin the regulation of a given mRNA could be better appreciatedby generating animals with specific disruption of this motif, touncouple mRNA expression from the control of IRPs.

Several outstanding questions on cellular iron metabolism,primarily related to the movement of iron across organelles, awaitfurther investigation. Future work will probably shed light on theroles of the siderophore 2,5-DHBA, Grx3 and Grx4, PCBP1 andthe E3 ubiquitin ligase FBXL5 in intracellular iron sensingand trafficking, and on the roles of ABC and SLC transporters andof FLVCR in the transport of haem and its precursors. The rapidlygrowing field of ISC biogenesis is expected to provide novellinks between cellular iron metabolism and utilization, and theidentification of iron-regulatory pathways involving microRNAsmay also be anticipated. Finally, characterization of the roles ofIRP1 and IRP2 in cancer biology may uncover novel functionsfor these proteins, not necessarily related to iron metabolism.

FUNDING

K.P. is funded by the Canadian Institutes for Health Research (CIHR) [grant number MOP-86514] and holds a Chercheur National career award from the Fonds de la Recherche enSante du Quebec (FRSQ).

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Received 8 November 2010/2 December 2010; accepted 3 December 2010Published on the Internet 24 February 2011, doi:10.1042/BJ20101825


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